The present invention relates to methods and materials for controlling gene silencing in plants, and various processes and products employing these methods and materials.
Co-suppression and Anti-sense Suppression of Endogenous Genes
It is known that stably-integrated transgenes (referred to as xe2x80x98STgenesxe2x80x99 or xe2x80x98intGENESxe2x80x99 below) which may be constitutively expressed may be used to suppress homologous endogenous (xe2x80x98HEgenesxe2x80x99) plant genes. This was discovered originally when chalcone synthase transgenes in petunia caused suppression of the endogenous chalcone synthase genes. Subsequently it has been described how many, if not all plant genes can be xe2x80x9csilencedxe2x80x9d by transgenes. Gene silencing requires sequence homology between the transgene and the gene that becomes silenced (Matzke, M. A. and Matzke, A. J. M. (1995), Trends in Genetics, 11: 1-3). This sequence homology may involve promoter regions or coding regions of the silenced gene (Matzke, M. A. and Matzke, A. J. M. (1993) Annu. Rev. Plant Physiol. Plant Mol. Biol., 44: 53-76, Vaucheret, H. (1993) C. R. Acad. Sci. Paris, 316: 1471-1483, Vaucheret, H. (1994), C. R. Acad. Sci. Paris, 317: 310-323, Baulcombe, D. C. and English, J. J. (1996), Current Opinion In Biotechnology, 7: 173-180, Park, Y-D., et al (1996), Plant J., 9: 183-194).
When coding regions are involved, the transgene able to cause gene silencing may have been constructed with a promoter that would transcribe either the sense or the antisense orientation of the coding sequence RNA. In at least one example the coding sequence transgene was constructed without a promoter (Van Blokland, R., et al (1994), Plant J., 6: 861-877).
Co-suppression of Transgenes
It has also become clear that gene silencing (gs) can account for some characteristics of transgenic plants that are not easily reconciled with conventional understanding of genetics. For example the wide variation in STgene expression between sibling lines with a Stgene construct is due in part to gene silencing: low expressers are those with a high level of gene silencing whereas high expressers are those in which gene silencing is absent or induced late in plant development. In this case there is no requirement for there to be an HEgene corresponding to the STgene (see e.g., Elamayan and Vaucheret (1996) Plant J., 9: 787-797.
Viral Resistance
In addition to observations of STgenes, gs has also been implicated in virus resistance. In these cases various factors including ectopic DNA interactions6, DNA methylation7, transgene expression level8 and double stranded RNA9 have been proposed as initiators of gene silencing.
Additionally in non-transgenic plants, it has been demonstrated that leaves which develop subsequently to systematic spread of a virus in a plant contain lower levels of virus than do symptomatic leaves. This resistance may be similar in nature to transgene-induced gene silencing (see e.g. Ratcliff et al (1997) Science, 276: 1558-1560).
Cytoplasmically Replicating Viral Constructs
Biosource Technologies, in WO 95/34668, have suggested the use of genetic constructions based on RNA viruses which replicate in the cytoplasm of cells to provide inhibitory RNA, either anti-sense or sense (xe2x80x9cco-suppressorxe2x80x9d) RNA. The constructs were used to inhibit a particular HEgene (phytoene desaturase). Cells were transfected with the cytoplasmically-replicating genetic constructions in which the RNA encoding region is specific for the gene of interest. The hybrid viruses spread throughout the plant, including the non-inoculated upper leaves (as verified by transmission electron microscopy). System-wide gene silencing was reported following transfection.
GB patent application 9703146.2, and PCT/GB98/00442, both filed in the name of John Innes Centre Innovations Limited, are hereby incorporated by reference. These applications, which were not published prior to the claimed priority date of the present application, discuss various constructs (xe2x80x98ampliconsxe2x80x99) which are intended to be stably integrated into the plant genome, and to generate cytoplasmically replicating constructs which are capable of eliciting gene silencing.
Silencing in Animals
Fire et al (1998) Nature 391: 806-811 (not published prior to the claimed priority date of the present application) discusses the use of RNA, particularly double-stranded RNA, to achieve silencing of endogenous genes and GFP-transgenes in nematodes. The demonstrated interference effect was apparently able to cross cell-boundaries.
Applications for Gene-silencing
In principle there is an enormous practical potential of gs for crop improvement. It is possible to silence genes conferring unwanted traits in the plant by transformation with transgene constructs containing elements of these genes. Examples of this type of application include gs of ripening specific genes in tomato to improve processing and handling characteristics of the harvested fruit; gs of genes involved in pollen formation so that breeders can reproducibly generate male sterile plants for the production of F1 hybrids; gs of genes involved in lignin biosynthesis to improve the quality of paper pulp made from vegetative tissue of the plant; gene silencing of genes involved in flower pigment production to produce novel flower colours; gene silencing of genes involved in regulatory pathways controlling development or environmental responses to produce plants with novel growth habit or (for example) disease resistance; elimination of toxic secondary metabolites by gene silencing of genes required for toxin production.
Gene silencing is also useful for investigating gene function in that it can be used to impose an intermediate or a null phenotype for a particular gene, which can provide information about the function of that gene in vivo.
A major complication in the practical exploitation of this phenomenon to date is the unpredictable and low occurrence of gene silencing. Therefore, it has not been realistic to attempt gene silencing in plants that are difficult to transform and for which it is difficult to produce many transformants. Similarly, it would be difficult to activate (and deactivate) gene silencing against several different traits or against several viruses in the same plant. Even with plants that are easy to transform the need to generate multiple lines limits the ease of exploitation of gene silencing.
The present inventors have now demonstrated a novel means of providing consistent, controlled, systemic gene silencing within a system, particularly a mature plant, which may (but is preferably not) a transgenic plant. This novel approach is clearly distinct from previously described approaches to gene silencing, for example, transwitch and antisense technologies, in that it describes a multicomponent system in which there is the potential to regulate the gene silencing spatially and optionally temporally.
The current invention is also distinct from the virus-induced gene silencing described previously by Biosource Technologies. In the current invention there is no absolute requirement that the transgenes conferring the gene silencing or their transcripts are able to replicate using viral components or through mechanisms that resemble virus replication, although in certain advantageous embodiments they may do so. Importantly, the systemic silencing of the invention does not require that the transgenes or their transcripts use virus-derived mechanisms to move between cells (e.g. xe2x80x98movement proteinsxe2x80x99 as they are termed in the art).
These movement proteins are encoded by most (probably nearly all) plant viruses. Movement proteins are normally recognised by mutation analysis of a viral genome. Mutation of a movement protein gene affects the ability of a virus to spread in the infected plant but does not affect the ability of the virus to replicate. Examples of viral movement proteins identified in this way include the 30 kDa protein of tobacco mosaic virus (Deom et al., 1987), the 25 kDa, 12 kDa and 8 kDa triple gene block proteins of potato virus X (FIG. 1C) (Angell and Baulcombe, 1995; Angell et al., 1996; Verchot et al., 1998) and the tubule-forming protein of cowpea mosaic virus (van Lent et al., 1991). Some viruses also encode movement proteins specifically for translocation of the virus through the phloem of the plant. Examples of these long distance movement proteins include the 2b protein encoded in cucumber mosaic virus (Ding et al., 1995) and the 19 kDa protein of tomato bushy stunt virus (Scholthof et al., 1995).
Until recently it has been considered that movement proteins open channels between plant cells and thereby mediate virus movement (Wolf et al., 1989). However it is now apparent that at least some of these proteins may also promote movement by suppression of a defence mechanism in the plant that blocks virus movement, which may itself be related to the gene silencing referred to hereinbefore. From these new findings, which are consistent with observations by Anandalakshmi et al. (1998) and Brigneti et al. (1998) [both in press] it is clear that movement proteins may be suppressors of gene silencing. Similarly the work of the present inventors suggests that certain proteins previously described only as pathogenicity proteins may also have a role in suppressing a gene silencing signal.
Thus it can be appreciated that stronger, systemic, gene silencing is obtained if transgene constructs for gene silencing do not also lead to expression of gene silencing by viral movement proteins or pathogenicity proteins, which are a fundamental part of the prior art systems which rely on the activity of vectors based on RNA-viruses. Such systems may be incapable of mediating a TIGS effect (see e.g. Dougherty, W. G, et al Molecular Plant-Microbe Interactions, 1994: 7, 544-552).
The novel gene silencing system of this invention was first demonstrated using transgenic N. benthamiana stably transformed with stably transformed with the gene for green fluorescent protein (designated stGFP).
The workers demonstrated that the expression of stGFP could be silenced by the transient presence of a GFP reporter gene (which was designated trGFP to distinguish it from the stGFP) using strains of Agrobacterium tumefaciens carrying binary Ti plasmid vectors or using direct infiltration. The silencing was systemic in nature, occurring remotely from the sites of infection or infiltration.
This approach has suggested the existence of a previously unknown signalling mechanism in plants that mediates systemic gene silencing. The signal of silencing is gene-specific and likely to be a nucleic acid that moves between cells.
A systemic, sequence-specific signal of gene silencing which is initiated by the transient presence (not stable integration) in part of a plant of foreign initiator nucleic acid or nucleic acid complex (termed hereinafter xe2x80x98fiNAxe2x80x99) which need not be capable of autonomous replication in the cytoplasm of a plant cell or movement from cell to cell, but which generates a signal which may be propagated systemically is an entirely novel and unexpected concept in plant biology. The observation has a number of important (industrially applicable) properties. These properties, and the characteristics of the fiNA required achieve them, will be discussed in more detail hereinafter.
The work of the present inventors, with hindsight, is consistent with data from other published experimental systems and could be a general feature of gene silencing in plants.
Thus transgenic petunia exhibiting transgene-induced silencing of the genes required for flower pigment biosynthesis were shown to exhibit unusual and irregular patterns of pigmentation. These can perhaps be explained by an extracellular signal rather than by cell lineage-dependent cues of gene silencing (see Jorgensen (1995) Science 268, 686-691). It should be stressed that in that work the gene silencing of an HEgene (CHS) was induced in the test plants using a chimeric STgene. Although the paper speculates about a 2 state system of gene silencing, no information is given about how to switch gene silencing on.
Work by a different group demonstrated chitinase gene silencing in non-clonal sectors of transgenic tobacco (see Kunz et al (1996) Plant J. 10, 4337-450.). This work demonstrated both the xe2x80x98selfxe2x80x99 inactivation of the expression of STgenes alone, plus inactivation of HEgenes by STgenes. The work also suggested that gene silencing was a post-transcriptional event. It was demonstrated that gene silencing occurred stochastically in progeny of transgenic plants but that xe2x80x98resettingxe2x80x99 to the non-silenced state occurred non-stochastically in developing seeds. These observations, plus the variegated pattern of silencing shown by some plants, demonstrated that the gene silencing phenotype was not merely a lineage event, but also highlighted the unpredictability of gene silencing. There is no suggestion in the paper of the use of fiNA to control gene silencing in non-silenced or xe2x80x98resetxe2x80x99 genes.
Palaqui et al, in The EMBO Journal (1997) V 16 No 15: pg 4738, demonstrated that grafting non-silenced scions onto gs-stock (co-suppressed ST and HE nitrate reductase genes,) imposes silencing on the scion. The scion had to contain the STgene, and the silencing was unidirectional and could occur through a wild-type stem xe2x80x98barrierxe2x80x99 in which HE nitrate reductase genes are present and function as signal transducing resident genes. Although a diffusible messenger is postulated, there is no mention of generating or employing this messenger other than by the use of grafts of already-silenced homozygous plant stock.
The systemic signal demonstrated by the present inventors is also consistent with recent findings that gene silencing is associated with induced natural defence against viruses. The signal could move in the plant ahead of the inducing virus so that anti-viral gene silencing could delay spread of the infection front (Ratcliff et al (1997) Science, 276: 1558-1560). The data below also suggests that in certain situations, viral proteins may act to inhibit this signal propagation.
The provision of the signalling mechanism and the novel means by which it can be activated (transient presence of fiNA) opens up a number of possibilities which will be discussed in more detail hereinafter; essentially the ability to conveniently control gene silencing systemically will be useful both in the investigation of gene function, and the production of gene silencing plants, as well as in the investigation of the mechanisms of gene silencing.
Particularly useful is the ability to rapidly and consistently impose, at will, gene silencing on HE or STgenes of known or unknown function in order to investigate their phenotype.
Although the systemic signal is not yet structurally characterised, a number of points about it can be made in the light of the present work. It is produced when fiNA is introduced in to a plant cell, particularly directly or indirectly into the cytoplasm, where the target gene or possibly a resident gene (as defined below) which is to be silenced is being transcribed, in the same plant cell, and there is sequence similarity between the coding regions of fiNA and target gene.
These findings suggest that a protein product, or the corresponding DNA or RNA, is a component of the signal. Of these, the protein product is the least plausible candidate because there is no mechanism known that explains how it could move systemically and specifically target the RNAs of the target. However, a nucleic acid-based signal could mediate sequence-specific gene silencing via a base-paired or triple helical structure with the target gene RNA (or the transcription product of homologous resident gene) as it moved between cells and tissues expressing that gene. Moreover, a nucleic acid could move in the plant, perhaps using the channels involved in virus or viroid movement. The demonstrated systemic spread of ST-GFP silencing (FIG. 2c) is consistent with this suggestion because it follows a course (FIGS. 2c, 2g) that is similar to the pattern of virus spread in an infected plant.
Thus in a first aspect of the invention there is disclosed a method for silencing a target nucleotide sequence (e.g. a gene) in a plant comprising transiently introducing (i.e. not via a stably integrated transgene) into the cytoplasm of cells of that plant in which the target sequence is present (and preferably being transcribed) a foreign initiator nucleic acid (fiNA) which is:
(i) incapable of movement from cell to cell, and
(ii) optionally incapable of autonomous replication, and
(iii) has sequence homology with the gene to be silenced.
This method is used for silencing a target gene in a first part of a plant comprising the steps of:
(a) transiently exposing a second part of the plant, remote from said first part, to a foreign initiator nucleic acid (fiNA) as described above such as to generate a gene silencing signal,
(b) causing or allowing the signal to be propagated to the second part of the plant such as to silence said target gene.
xe2x80x9cCausing or allowingxe2x80x9d in this sense implies, in particular, that the construct giving rise to the fiNA (and hence signal) does not encode proteins which would block the signal e.g. movement proteins such as those which permit viral movement from cell to cell.
Thus the present inventors have demonstrated for the first time Transiently Induced Gene Silencing (or xe2x80x98TIGSxe2x80x99). They have further demonstrated that a signal capable of propagating gene silencing can be initiated in a second part of the plant to induce silencing of a gene in the first.
Generally speaking, TIGS can be considered as having three phases:
(i) initiation of a gene silencing signal by the transient presence of fiNA in the cytoplasm of plant cells, which is described in more detail below,
(ii) translocation of a gene silencing signal (though not the fiNA itself) through tissues of the plant, which is facilitated by the expression of a HE gene or a ST gene with homology to the target gene in those tissues,
(iii) maintenance of the gene silencing signal within the cells of the plant, which may be remote from those which were initially, transiently, exposed to the fiNA.
The various different features of TIGS will now be discussed in more detail:
xe2x80x9cSilencingxe2x80x9d in this context is used to refer to suppression of expression of the (target) gene. It does not necessarily imply reduction of transcription, because gene silencing is believed to operate in at least some cases post-transcriptionally. The degree of reduction may be so as to totally abolish production of the encoded gene product (yielding a null phenotype), but more generally the abolition of expression may be partial, with some degree of expression remaining (yielding an intermediate phenotype). The term should not therefore be taken to require complete xe2x80x9csilencingxe2x80x9d of expression. It is used herein where convenient because those skilled in the art well understand this.
The xe2x80x9csystemicxe2x80x9d silencing means that the target gene is silenced via a signal which is translocated substantially throughout the tissues of a plant (though certain tissues may not be silenced e.g. meristematic tissues, as discussed in more detail below).
The xe2x80x9ctargetxe2x80x9d gene (ie the gene to be silenced or the silenced gene) in the present invention may be any gene of interest. As discussed below it will share homology with the fiNA. In particular it may be a homologous endogenous gene (HEgene) or a stably transformed homologous transgene (STgene, as with the stGFP used above).
More than one target gene (e.g. a gene family) may be targeted simultaneously provided that they all share homology with the fiNA.
As will be discussed in more detail hereinafter, in certain aspects of the invention the identity or phenotype of the gene may be unknownxe2x80x94and indeed TIGS may be used to identify it.
The xe2x80x9cfiNAxe2x80x9d, which may be either DNA or RNA, may be synthetic (ie man made) or naturally occurring nucleic acid sequence which is a homolog of the target gene or it may be a copy of all or part of the target gene in sense or antisense orientation. It may be double or single stranded, for instance it may consist of antisense (double stranded) RNAs.
It should be stressed that, unlike RNA viral-based vectors used to effect gene silencing in the art (e.g Biosource Technologies, in WO 95/34668) the fiNA itself lacks sequences which permit movement from plant cell to plant cell, and optionally allow replication in the cytoplasm of plant cells (i.e. fiNA need not be capable of autonomous replication in the cell).
Unlike the amplicons of PCT/GB98/00442 (which may optionally lack such movement sequences) fiNA is not generated by a stably integrated transgene in the plant.
Thus the crucial elements of the fiNA which give the potential for signal initiation are that:
(i) it is foreign to the plant, or is at least recognised as being foreign, possibly after interacting with existing nucleic acids in the plant,
(ii) it shares homology with all or part of the target gene (coding or non-coding strand),
(iii) it cannot move from plant cell to plant cell (more particularly, does not comprise sequence encoding movement proteins or other pathogenicity proteins which would interfere with the signal) and optionally it cannot replicate autonomously in plant cell cytoplasm.
The term xe2x80x9cforeignxe2x80x9d is used broadly to indicate that the fiNA has been introduced into the cells of the plant or an ancestor thereof, possibly using recombinant DNA technology, but in any case by human intervention. Put another way fiNA will be non-naturally occurring in cells in to which it is introduced. For instance the fiNA may comprise a coding sequence of or derived from a particular type of plant cell or species or variety of plant, or virus, placed within the context of a plant cell of a different type or species or variety of plant. Alternatively the fiNA may be derived from the plant genome but is present in xe2x80x9cunnaturalxe2x80x9d cellular or chromosomal locations, or lacks certain features of the authentic endogenous sequence (gene or transcript). A further possibility is for the fiNA to be placed within a cell in which it or a homolog is found naturally, but wherein the fiNA is linked and/or adjacent to nucleic acid which does not occur naturally within the cell, or cells of that type or species or variety of plant, such as operably linked to one or more regulatory sequences, such as a promoter sequence, for control of expression.
Regarding the xe2x80x9chomologyxe2x80x9d of the fiNA, the complete sequence corresponding to the transcribed sequence need not be used to effect gene silencing, as is clear from the prior art studies (which albeit did not use fiNA as described herein or provide TIGS). For example fragments of sufficient length may be used. It is a routine matter for the person skilled in the art to screen fragments of various sizes and from various parts of the coding or non-coding sequence of the target gene to optimise the level of gene silencing, for instance using systems based on the GFP system described later. It may be advantageous to include the initiating methionine ATG codon of the target gene, and perhaps one or more nucleotides upstream of the initiating codon. A further possibility is to target a conserved sequence within a target gene, e.g. a sequence that is characteristic of one or more target genes in order to silence several genes which comprise the same or similar conserved sequence.
A fiNA may be 300 nucleotides or less, possibly about 200 nucleotides, or about 100 nucleotides. It may be possible to use oligonucleotides of much shorter lengths, 14-23 nucleotides. Longer fragments, and generally even longer than 300 nucleotides are preferable where possible if the fiNA is produced by transcription or if the short fragments are not protected from cytoplasmic nuclease activity.
It may be preferable that there is complete sequence identity between the fiNA and a relevant portion of the target sequence, although total complementarity or similarity of sequence is not essential. One or more nucleotides may differ in the targeting sequence from the target gene. Thus the fiNA of the present invention may correspond to the wild-type sequence of the target gene, or may be a mutant, derivative, variant or allele, by way of insertion, addition, deletion or substitution of one or more nucleotides, of such a sequence.
The fiNA need not include an open reading frame or specify an RNA that would be translatable. There may be a TIGS signal even where there is about 5%, 10%, 15%, 20% or 30% or more mismatch between the fiNA and the corresponding homologous target sequence. Sequence homology (or xe2x80x98identityxe2x80x99 or xe2x80x98similarityxe2x80x99xe2x80x94the terms are used synonymously herein) may be assessed by any convenient method e.g. it may determined by the TBLASTN program, of Altschul et al. (1990) J. Mol. Biol. 215: 403-10, which is in standard use in the art.
Regarding translocation of the TIGS signal, as described above this is generated when the cells of the plant are transiently exposed to the fiNA, and the translocating tissues comprise, and preferably transcribe (though not necessarily express) the target gene or another xe2x80x98resident genexe2x80x99 sharing homology with the target gene and the fiNA for the gene silencing signal to be transmitted through such tissues. However it may not be necessary for all of the translocating tissues to transcribe the genexe2x80x94as shown in the Examples below, the signal may be xe2x80x98relayedxe2x80x99 between expressing cells.
The resident gene, which is discussed in more detail below, may be either endogenous or exogenous to the plant. The term xe2x80x98homologyxe2x80x99 in relation to the resident gene is used in the same way as it is used in relation to the fiNA/target gene above. In this case the crucial element is that the homology be sufficient to allow signal generation and/or propagation. As described above the homology will preferably be at least 70%, more preferably at least 75%, more preferably at least 80%, more preferably at least 85%, more preferably at least 90% or most preferably more than 95%.
The advantage of using an STgene as a resident gene is that its transcription may be more readily controlled (if desired) than a target gene which is an HEgene, as is discussed in more detail in relation to facilitating signal propagation below.
The xe2x80x9ctransient exposurexe2x80x9d of the second part of the plant to the fiNA may be achieved by any convenient method. Essentially the fiNA should be introduced directly or indirectly (e.g. exposure of a fiNA produced in the nucleus from locally present foreign nucleic acid) into the cytoplasm of cells of the second part of the plant.
Known methods of introducing nucleic acid into plant cells include use of a disarmed Ti-plasmid vector carried by Agrobacterium exploiting its natural gene transfer ability (EP-A-270355, EP-A-0116718, NAR 12(22) 8711-8721 (1984), particle or microprojectile bombardment (U.S. Pat. No. 5,100,792, EP-A-444882, EP-A-434616) microinjection (WO 92/09696, WO 94/00583, EP 331083, EP 175966, Green et al. (1987) Plant Tissue and Cell Culture, Academic Press), electroporation (EP 290395, WO 8706614) other forms of direct DNA uptake (DE 4005152, WO 9012096, U.S. Pat. No. 4,684,611), liposome mediated DNA uptake (e.g. Freeman et al. Plant Cell Physiol. 29: 1353 (1984)), or the vortexing method (e.g. Kindle, PNAS U.S.A. 87: 1228 (1990d) Physical methods for the transformation of plant cells are reviewed in Oard, 1991, Biotech. Adv. 9: 1-11.
Preferably fiNA is introduced by microprojectile bombardment with gold particles. Vacuum infiltration or injection of agrobacterium or direct uptake mediated by carborundum powder, whiskers (see Frame et al, Plant J 1994, 6: 941-948) or electroporation.
Various Embodiments Will Now Be Exemplified
Introduction of fiNAxe2x80x94Initiation of the Signal
As described above fiNA may be introduced directly as naked DNA, or it may be transcribed from nucleic acid introduced into (but not stably integrated throughout) a plant. It should be stressed that although the fiNA must be located in the cytoplasm of the cell, there is no requirement that the fiNA be transcribed in the cell; thus there is no need for the fiNA to incorporate a promoter region in order to initiate the gene silencing signal or for it to be introduced into the cytoplasm via the nucleus.
In a further embodiment it may be possible to use a viral or other extrachromosomal expression vector (which may or may not include translation signals) e.g. a viral-based vector, encoding the fiNA, and a replicase, but lacking transmissive elements (e.g. movement proteins or other pathenogenicity proteins) which could inhibit the generation of a signal which can move beyond the infected parts of the plant, or be sustained within the plant after initial introduction. However viruses, particularly those which are transmissible, may be undesirable for other reasons e.g. safety, resistance etc.
In a further embodiment it may be achieved by transiently (e.g. locally) initiating the transcription of a fiNA-encoding sequence which is present in the cells, possibly the nucleus or the genome, of the second part of the plant.
This may be achieved by the use of Ti-based binary vectors (cf. use of the trGFP described below). Generally speaking, those skilled in the art are well able to construct vectors and design protocols for transient recombinant gene transcription. For further details see, for example, Molecular Cloning: a Laboratory Manual: 2nd edition, Sambrook et al, 1989, Cold Spring Harbor Laboratory Press.
Optionally transcription of the fiNA may be placed under the control of an activating agent, for instance using an inducible promoter.
The term xe2x80x9cinduciblexe2x80x9d as applied to a promoter is well understood by those skilled in the art. In essence, transcription under the control of an inducible promoter is xe2x80x9cswitched onxe2x80x9d or increased in response to an applied stimulus. The nature of the stimulus varies between promoters. Some inducible promoters cause little or undetectable levels of transcription (or no transcription) in the absence of the appropriate stimulus. Other inducible promoters cause detectable constitutive expression in the absence of the stimulus. Whatever the level of expression is in the absence of the stimulus, expression from any inducible promoter is increased in the presence of the correct stimulus.
One example of an inducible promoter is the GST-II-27 gene promoter which has been shown to be induced by certain chemical compounds which can be applied to growing plants. The promoter is functional in both monocotyledons and dicotyledons. It can therefore be used to control gene expression in a variety of genetically modified plants, including field crops such as canola, sunflower, tobacco, sugarbeet, cotton; cereals such as wheat, barley, rice, maize, sorghum; fruit such as tomatoes, mangoes, peaches, apples, pears, strawberries, bananas, and melons; and vegetables such as carrot, lettuce, cabbage and onion. The GST-II-27 promoter is also suitable for use in a variety of tissues, including roots, leaves, stems and reproductive tissues. Other example inducible promoters are well known to those skilled in the art, the choice of which will be determined by the convenience of using the inducing agent in the particular application being carried out.
Another suitable promoter may be the DEX promoter (Plant Journal (1997) 11: 605-612).
In this embodiment the activating agent can be applied locally to one or more regions of the plant in which the fiNA-encoding construct has been introduced (the xe2x80x98second partxe2x80x99) in order to achieve the remote silencing of other (xe2x80x98first partxe2x80x99).
In a most preferred aspect, the fiNA may be introduced as a construct corresponding to a truncated xe2x80x98ampliconxe2x80x99 of GB 98/00442. This will generally comprise:
(i) a plant promoter
(ii) a nucleic acid sequence operably linked to that promoter, said sequence encoding an RNA-dependent replicase, and further encoding fiNA, which is itself operably linked to a sub-genomic promoter capable of being recognised by said replicase, such that the fiNA is capable of autonomous cytoplasmic replication, with the proviso that the nucleic acid sequence does not encode active viral movement proteins (plus optionally pathogenicity proteins) which would otherwise inhibit the TIGS signal from spreading systemically in the plant into which the construct is introduced.
By xe2x80x9creplicasexe2x80x9d is meant, where appropriate, all the required components to give replicase function. The construct does not encode xe2x80x9cactive movement proteinsxe2x80x9d in the sense that, although a movement proteins may be encoded, they are not functional e.g. because one or more has been deleted or modified.
Propagation and Maintenance of the Signal Through the Plant
The advantage of achieving systemic gene silencing using transient activation or introduction of fiNA in a localised area (e.g. by application of a specific agent) is that there is no requirement for the inducing agent of fiNA to be translocated within the tissues of the plant or be applied to all parts of the plant. Once initiated the signal can induce gene silencing in remote parts of the plant. This gene silencing is stable and persists even after the fiNA ahs been removed.
By xe2x80x9cremotexe2x80x9d is meant the first and second parts of the plant are spatially separated, although obviously connected via the plant tissues. It may be advantageous if the first part of the plant is above the level of the second, or if the route corresponds to the xe2x80x98source-sinkxe2x80x99 movement of photosynthetic products from regions in which they are concentrated to regions of use. The observations described in the Examples suggest that signal movement mimics in some respects viral or viral-vector movement. It should be stressed, however, that neither the signal of the present invention, nor the fiNA used to initiate it, are viruses, for instance mobile, cytoplasmatically replicable vectors.
It should also be stressed that the part of the plant in which the target gene is to be silenced may encompass all, or almost all, of that part of the plant which is not directly exposed to the fiNA i.e. systemic silencing.
Thus in one embodiment of this aspect, the target gene is silenced systemically in the plant tissues i.e. in the first and second parts of the plant and the tissues between them, (cf. the stGFP described below).
It may not be necessary for all the cells in these tissues to transcribe the target gene, as detailed in the Examples.
Alternatively, some or all of the cells of the connecting plant tissues will comprise a resident gene, the transcription (though not necessarily expression) facilitates the propagation of the signal.
By xe2x80x9cresident genexe2x80x9d is meant a gene (endogenous or exogenous) which is homologous to the target gene and homologous to the fiNA such as to facilitate transduction of the TIGS signal.
Thus in a second embodiment of this aspect, the target gene is transcribed only in a second, remote, part of the plant (e.g. it is expressed in a tissue specific manner), but a resident gene which is homologous to the target gene is present and preferably transcribed in the plant tissues in the second part of the plant and/or the tissues between the first and second parts of the plant. Presence or preferably transcription of this resident gene may thus serve to cause or allow signal propagation.
This embodiment permits control of tissue specific target genes. The resident gene serves to assist systemic spread of the signal. The systemic spread of the signal can thus be controlled at an additional level to the direct control of the fiNA exposure, providing further temporal and spatial control over gene silencing:
By regulating the transcription of the resident gene in the cells carrying the TIGs signal, it will be possible to determine whether gene silencing in the first part of the plant is activated effectively, or to affect the tissue specificity of gene silencing.
Transcription of a resident (STgene) may be altered by use of an inducible promoter, such as is described above in relation to the fiNA.
It will be apparent from the foregoing that the invention embraces methods of controlling gene silencing in plants by manipulating the presence or transcription of the fiNA or the propagation of the signal. e.g. by controlling the presence or absence of an activating agent which induces transcription of a resident gene. Physical methods for manipulating the resident gene expression are also envisaged. For instance grafts of tissue between the different parts of the plant which are either permissive (i.e. contain cells having the resident gene) or non-permissive (cells don""t have the resident gene) can be used to control translocation of the signal.
Selected Applications for TIGS
In embodiments of the present invention which have been experimentally exemplified as described below for illustrative and non-limiting purposes only, the transiently introduced gene encoding the fiNA that determined the target of gene silencing was the gene encoding the jellyfish green fluorescent protein GFP (Chalfie et al. (1994) Science 263: 802-805). This was used to silence a stably integrated GFP transgene.
Any other ST- or HEgene of a plant, or STgene of animal, fungal, bacterial or viral origin may be a target gene provided that the fiNA contains a corresponding homologous sequence.
In one aspect of the present invention, the target gene may be of unknown phenotype, in which case the TIGS system may be employed to analyse the phenotype by generating a systemic (or widespread) null (or nearly null) phenotype.
Thus a further aspect of the invention comprises a method of characterising a target gene comprising the steps of:
(a) silencing the target gene in a part or at a certain development stage of the plant using the TIGS system described above,
(b) observing the phenotype of the part of the plant in which or when the target gene has been silenced.
Preferably the gene is silenced systemically. Generally the observation will be contrasted with a plant wherein the target gene is being expressed in order to characterise (i.e. establish one or more phenotypic characteristics of) the gene.
There are several advantages of the current method over alternative methods in which the targeted gene is inactivated by insertional or other mutagenic procedures or in which gene silencing is uncontrolled. The advantage over mutagenic procedures applies when there is more than one homologous gene carrying out the role of the target gene. Mutagenic procedures will not normally reveal a phenotype in that situation. A second situation where the current invention has advantage over both mutagenic and unregulated gene silencing procedures applies when the target gene has a lethal phenotype. The controllable attribute of the gene silencing will allow the phenotype of such genes to be investigated and exploited more efficiently than using the alternative methods available prior to the disclosure of the current invention.
This aspect is particularly useful given the significant amount of sequence data currently being generated in genomics projects which is unassigned in terms of function or phenotype. Thus even if the gene exerts its effects only in particular tissues, this may be detectable without having to ensure that a virus has permeated the entire plant (as in Biosource Technologies, WO 95/34668).
Nor, for the identification of HE genes, would it be necessary to try and generate a transgenic plant in which gene silencing is already activated to observe the effect.
In a further aspect there is disclosed a method of altering the phenotype of a plant comprising use of the TIGS method.
Traits for which it may be desirable to change the phenotype include the following: colour; disease or pest resistance; ripening potential; male sterility.
For instance male sterile plants are required for production of hybrid seed. To propagate the male sterile lines it is necessary to restore male fertility. In the examples in which male sterility is induced by a transgene it would be possible to restore male fertility by controlled silencing of the transgene using the approach described above.
Many genes have multiple roles in development. They may be required, for example, in embryo development and in the development of organs or tissues in the mature plant. Obviously it would not be possible to silence these genes unless the silencing system could be controlled so that it is not active in embryo development. The system described here could be used to provide that control.
Other traits will occur to those skilled in the art. In each case the only necessity is that sufficient is known about the target gene(s) to devise suitable fiNA, which may of course be optimised without burden to achieve the desired effect. If the target gene is not expressed systemically, then it may be necessary to produce a transgenic plant wherein a resident STgene is transcribed systemically in order to allow signal propagation. The fiNA can then be used to initiate the signal.
The production of transgenic plants is now very well known to those skilled in the art, as evidenced by the various reported methods some of which are recorded in non-prior published GB patent application 9703146.2 in the name of John Innes Centre Innovations Limited, the content of which is incorporated herein by reference.
In a further aspect of the present invention there is disclosed a method for producing a systemic gene silencing signaling agent in a plant, which is capable of silencing a target gene comprising causing or allowing the transient exposure of a part of the plant expressing said target gene or a homolog thereof to a fiNA.
The systemic gene silencing signaling agent is characterised in that it
(a) comprises nucleic acid,
(b) is capable of mediating sequence-specific gene silencing of a target gene,
(c) it is obtainable by transient exposure of a plant cell transcribing said target gene or a homolog thereof to a fiNA,
(d) is capable of moving between a first and second part of the plant, said parts being connected by cells comprising, and preferably transcribing said target gene or a homolog thereof, which movement is inhibited my movement or pathogenicity proteins as discussed above.
The various nucleic acids of the present invention may be provided isolated and/or purified (i.e. from their natural environment), in substantially pure or homogeneous form, or free or substantially free of other nucleic acid. Nucleic acid according to the present invention may be wholly or partially synthetic. The term xe2x80x9cisolatexe2x80x9d encompasses all these possibilities.
Also embraced by the present invention is a transgenic plant comprising a target gene which has been systemically silenced using TIGS.
The present invention may be used in plants such as crop plants, including cereals and pulses, maize, wheat, potatoes, tapioca, rice, sorgum, millet, cassava, barley, pea and other root, tuber or seed crops. Important seed crops are oil seed rape, sugar beet, maize, sunflower, soybean and sorghum. Horticultural plants to which the present invention may be applied may include lettuce, endive and vegetable brassicas including cabbage, broccoli and cauliflower, and carnations and geraniums. The present invention may be applied to tobacco, cucurbits, carrot, strawberry, sunflower, tomato, pepper, chrysanthemum, poplar, eucalyptus and pine.
The present invention will now be illustrated and exemplified with reference to experimental results and the accompanying Figures. Further aspects and embodiments of the present invention, and modifications of those disclosed herein, will be apparent to those skilled in the art. All documents mentioned anywhere herein are incorporated by reference.